METHODS AND COMPOSITIONS RELATED TO CALCIUM HOMEOSTASIS

AND FABRY DISEASE

BACKGROUND OF THE INVENTION [0001) This application claims the benefit of priority to U.S. Provisional Patent

Application Serial No. 61/763,840 filed February 12, 2013 hereby incorporated by reference in its entirety.

1. Field of the Invention

[0002] The present invention relates generally to the field of medicine. More particularly, it concerns the use of calcium cycling modulators to treat arrhythmias and related heart conditions in Fabry disease patients.

2. Description of Related Art

[0003] Fabry disease is an X-linked lysosomal storage disorder, caused by insufficient activity of a-galactosidase A (oc-Gal A). As a result of enzyme deficiency glycosphingolipids (GSLs) with terminal a-D-galactosyl moieties, primarily globotriaosylceramide (Gb3), accumulate in multiple organs (Brady, et ah, 1967). Arrhythmias are one of the most prominent cardiac manifestations of Fabry disease, which can cause sudden death and precipitate heart failure in the patients. These include bradycardia, various degrees of atrioventricular blocks and various types of ectopic atrial and ventricular rhythms (Schiffman, 2009; O'Mahony, et al, 201 1 ). The pathogenesis of arrhythmias in Fabry disease remains unclear. Enzyme replacement therapy, the only available specific treatment for Fabry disease, has very limited effect on preventing or correcting arrhythmias.

[0004] Currently the only approved specific treatment for Fabry disease patients is enzyme replace therapy (ERT). ERT in Fabry disease has very limited effect, especially in preventing and treating cardiac arrhythmias and other cardiac complications. Another treatment called pharmacological chaperon is in development for Fabry disease patients who have amendable mutations. Both the ERT and chaperon studied are based on the concept of the supplement of the deficient enzyme to the patient or stabilizing the mutant enzyme to increase enzyme activity. SUMMARY OF THE INVENTION

[0005] Methods and compositions are provided for treating arrhythmia or treating a patient with or at risk for Fabry disease. Additional methods and compositions can be used for treating patients with suspected of having arrhythmia or brachycardia or having symptoms of arrhythmia or brachycardia, or patients at risk for arrhythmia or brachycardia. Methods and compositions concern regulating calcium homeostasis in cardiac cells. In certain embodiments, calcium homeostasis is restored in cardiac cells, such as cardiomyocytes.

[0006] Some embodiments concern a pharmaceutical composition comprising a calcium cycling modulator, wherein the calcium cycling modulator restores calcium homeostasis in the cardiac cells. In some aspects, the calcium cycling modulator is an L-type calcium channel antisense nucleic acid. In certain embodiments, the L-type calcium channel antisense nucleic acid is 8 to 50 nucleotides in length. Further embodiments include an L- type calcium channel antisense nucleic acid that is partly or fully double stranded. Some embodiments involve an antisense nucleic acid that comprises DNA. It is contemplated that the nucleic acid may include one or more modified nucleic acid residues. In other embodiments, the calcium cycling modulator is a CASQ2 polypeptide or nucleic acid encoding a CASQ2 polypeptide. In specific embodiments the nucleic acid is an expression vector or expression construct. In certain embodiments, the expression vector is a viral vector. [0007] Other embodiments include a number of different methods that may or may not use the pharmaceutical compositions described above or elsewhere.

[0008] In some embodiments there are methods of treating a patient with Fabry disease comprising administering to the patient an effective amount of a composition comprising a calcium cycling modulator, wherein the calcium cycling modulator restores calcium homeostasis in cardiac cells of the patient. In some embodiments there are methods of treating a patient with Fabry disease comprising administering to the patient an effective amount of a composition comprising a calcium cycling modulator, wherein the calcium cycling modulator restores calcium homeostasis in other systems (apart from the heart) of the patient. Other methods include treating arrhythmia in a patient with Fabry disease comprising administering to the patient an effective amount of a composition comprising a calcium cycling modulator, wherein the calcium cycling modulator restores calcium homeostasis in the cardiac cells. Further embodiments include methods of treating brachycardia in a Fabry disease patient comprising administering to the patient an effective amount of a composition comprising a calcium cycling modulator. Other embodiments include methods of decreasing Ryanodine receptor 2 hyperphosphorylation (e.g., by use of a phosphatase) in a Fabry disease patient, comprising administering to the patient an effective amount of a composition comprising a calcium cycling modulator. Additional embodiments include methods of increasing phospholamban in a Fabry disease patient comprising administering to the patient an effective amount of a composition comprising a calcium cycling modulator. In other embodiments, the calcium cycling modulator is a phospholamban polypeptide or nucleic acid encoding a phospholamban polypeptide. In specific embodiments the nucleic acid is an expression vector or expression construct. In certain embodiments, the expression vector is a viral vector. Other embodiments include methods of restoring calcium homeostasis in a patient in need thereof comprising administering a calcium cycling modulator.

[0009] Patients may have been diagnosed or identified as in need of treatment in some embodiments. In certain embodiments, a patient is determined to be in need of restoration of calcium homeostasis.

[0010] In some embodiments, the calcium cycling modulator restores calcium homeostasis by preventing calcium overload and/or reducing sarcoplasmic reticulum (SR) calcium leak. In further embodiments, the calcium cycling modulator inhibits calcium channel activity or expression. In particular embodiments, the calcium cycling modulator selectively inhibits calcium channel activity or expression. In some aspects, the calcium cycling modulator inhibits L-type calcium channel activity or expression. It is contemplated that the calcium cycling modulator is a non-dihydropyridine in some embodiments. Additional embodiments concern a calcium cycling modulator that inhibits L-type calcium channel activity. In particular embodiments, the calcium cycling modulator is verapamil or diltiazem, or a salt or prodrug thereof. In some embodiments, the calcium cycling modulator is not diltiazem. In other embodiments, the calcium cycling modulator is mibefradil, bepridil, fluspirilene, or fendiline, or a salt or prodrug thereof. In some embodiments, the calcium cycling modulator is an L-type calcium channel antisense nucleic acid, as described herein. [0011 J Methods include a calcium cycling modulator stabilizes Ryr2 channels or increases calsequestrin 2 (CASQ2) activity or expression. In some cases, the calcium cycling modulator increases CASQ2 activity or expression. In some cases, the calcium cycling modulator increases CASQ2 expression. In some cases, the calcium cycling modulator increases phospholamban activity or expression.

[0012] Certain embodiments involve a calcium cycling modulator that is 1 ,4- benzothiazepine analogue JTV519 ( 201), JTV519 analogue (S I 07), RyR2 blocking agent tetracaine or CaMKII inhibitor K.N-93. In some embodiments, the calcium cycling modulator is BAPTA or EDTA.

[0013] Some methods involve a patient who has previously been administered enzyme replacement therapy. Other methods comprise treating the patient with enzyme replacement therapy. In further embodiments, the calcium cycling modulator is administered multiple times. It may be administered multiple times per day. It may be administered intravenously, intraarterial ly, intraperitoneally, intradermal ly, intramuscularly, subcutaneously, intranasally, intratracheally, intraspinally, intracranial ly, orally, or by infusion.

[0014] Methods may include evaluating the patient for cardiac arrhythmia or for Fabry's disease or for brachycardia. A patient may be further monitored for brachycardia or cardiac arrhythmia following treatment.

[0015] Other embodiments include the use of a composition described herein for the treatment of arrhythmia in a patient with Fabry disease.

[0016] A dose may be administered on an as needed basis or every 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 18, or 24 hours (or any range derivable therein) or 1 , 2, 3, 4, 5, 6, 7, 8, 9, or times per day (or any range derivable therein). A dose may be first administered before or after signs of arrhythmia are exhibited or felt by a patient or after a clinician evaluates the patient for an arrhythmia. In some embodiments, the patient is administered a first dose of a regimen 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12 hours (or any range derivable therein) or 1 , 2, 3, 4, or 5 days after the patient experiences or exhibits signs or symptoms of an arrhythmia (or any range derivable therein). The patient may be treated for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days (or any range derivable therein) or until symptoms of an arrhythmia have disappeared or been reduced or after 6, 12, 18, or 24 hours or 1 , 2, 3, 4, or 5 days after symptoms of arrhythmia have disappeared or been reduced. |0017) As used herein the specification, "a" or "an" may mean one or more. As used herein in the claim(s), when used in conjunction with the word "comprising", the words "a" or "an" may mean one or more than one.

(0018] The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or." As used herein "another" may mean at least a second or more.

[0019] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

[0020] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0022] FIG. 1A-1G: Generation of human iPSC from Fabry patients and healthy controls. Dermal fibroblasts (A) from Fabry patients and healthy controls are used for generation of iPSC (B,C). The established iPSC lines are positive for TRA- 1 -60 (D), SSEA-4 € and Nanog (F). The mutations in the GLA gene of Fabry patients iPSC are confirmed by DNA sequencing (G).

[0023] FIG. 2A-2G: Cardiac differentiation. Cardiac cells (A) differentiated from Fabry patient and control iPSC express cardiac cell-specific markers. Troponin T, GATA-4 (B) and sarcomeric a-Actinin (C-F). Quantitative mRNA expression analysis (G) show cardiomyocytes from Fabry and healthy controls express similar level of TNNT2, troponin T type 2 (cardiac); MYH6, myosin, heavy chain 6, cardiac muscle, alpha and NKX2.5, NK2 homeobox 5.

[0024] FIG. 3A-3B: GSLs accumulation. Gb ₃ immunostaining in untreated (A) and a-Gal A treated (B) Fabry patient iPSC-derived cardiomyocytes.

[0025] FIG. 4A-4B: "Bradycardia" in cardiomyocytes differentiated from Fabry patient iPSC. Representative differentiated beating clusters of cardiomyocytes are shown in A. Spontaneous beating rates of the beating clusters from Fabry patients are significantly slower than those from the healthy controls (B). *P < 0.01. [0026] FIG. 5: Arrhythmia in donor Fabry disease patient. E G recording of

Fabry patient showed abnormal EKG: marked sinus bradycardia with 1 ^st degree A-V block, right bundle branch block, left anterior fascicular block, incomplete trifascicular block, lateral infact. Vent rate: 48 BPM; PR interval: 232 ms; QRS duration 180 ms; QT/QTc: 478/427 ms.

[0027] FIG. 6: Quantitative RT-PCR analysis of the expression levels of calcium regulatory proteins in Fabry patient and control iPSC-derived cardiomyocytes. *p <

0.05

[0028] FIG. 7A-7C: Ca2+ imaging study in Fabry patients and healthy control cardiomyocytes. (A) Ventricular cardiomyocytes were identified by Tomato red (arrow). (B) Ca2+ transients were recorded. (C) The spontaneous firing rates, amplitude, upstroke and decay velocities of the Ca2+ transient in Fabry patient and healthy control cardiomyocytes were measured.

[0029J FIG. 8A-8B: Western blot analysis of RyR2 and PLN. (A) The phosphorylated and total RyR2 and PLN were measured in the Fabry patient iPSC-derived cardiomyocyte clusters by Western blot. (B) Western blot analysis of RyR2 and PLN in the Fabry mouse heart tissues.

[0030] FIG. 9A-9B: Effect of verapamil on the beating rates of Fabry patient iPSC-derived cardiac clusters. (A) MEA tracing of diseased cardiac clusters treated with low dose verapamil. (B) Beating rates of diseased and normal control cardiac clusters treated with verapamil. Data were presented as mean ± s.e.m. DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

I. The Present Invention

[0031] As discussed above, the compositions and methods of using these compositions can treat a subject (e.g., Fabry disease subject with calcium homeostasis pathology) having, suspected of having, or at risk of developing cardiac manifestations of Fabry disease.

II. Fabry Disease

[0032] Fabry disease, also known as Fabry's disease, Anderson-Fabry disease, angiokeratoma corporis diffusum and alpha-galactosidase A deficiency is a rare genetic lysosomal storage disease, inherited in an X-linked manner. Fabry disease can cause a wide range of systemic symptoms, including pain, renal involvement, cardiac manifestations, dermatological manifestations, ocular manifestations, Fatigue, neuropathy (in particular, burning extremity pain), cerebrovascular effects leading to an increased risk of stroke, tinnitus, vertigo, nausea, inability to gain weight, chemical imbalances, and diarrhea. [0033] Fabry disease is a form of sphingolipidosis, as it involves dysfunctional metabolism of sphingolipids. A deficiency of the enzyme alpha galactosidase A (a-GAL A, encoded by GLA) due to mutation causes a glycolipid known as globotriaosylceramide (abbreviated as Gb3, GL-3, or ceramide trihexoside) to accumulate within the blood vessels, other tissues, and organs. This accumulation leads to an impairment of their proper function. [0034] Cardiac complications occur in Fabry disease subjects when glycolipids build up in different heart cells. Heart related effects worsen with age and may lead to increased risk of heart disease. Hypertension (high blood pressure) and cardiomyopathy are commonly observed. Arrhythmias and cardiac hypertrophy are the most prominent cardiac manifestations of Fabry disease, which can cause sudden death and precipitate heart failure in the patients.

III. Methods of restoring calcium homeostasis in cardiac cells

[0035] In certain embodiments of the current methods, cardiac cells of a Fabry disease subject are treated by administering a composition to affect calcium modulation in cardiac cells and restore calcium homeostasis. In specific embodiments, the composition to affect calcium modulation comprises a calcium cycling modulator. A calcium cycling modulator may operate in specific embodiments by preventing calcium overload or reducing calcium leak from the sarcoplasmic reticulum.

[0036] In certain embodiments, a calcium cycling modulator inhibits calcium channel activity. In specific embodiments, calcium channel activity is selectively inhibited. In yet other specific embodiments, the calcium channel is an L-type calcium channel. In certain embodiments, the calcium modulator that inhibits an L-type calcium channel is a non- dihydropyridine. In certain aspects a dihydropyridine belongs to one of the phenylalkylamines or benzothiazepine groups. In certain aspects the calcium cycling modulator is Verapamil, Gallopamil, Fendiline or Diltiazem. In other aspects the calcium cycling modulator is mibefradil, bepridil, fluspirilene, or fendiline.

[0037] Administration of drugs such as Verapamil, Gallopamil, Fendiline, Diltiazem, mibefradil, bepridil, fluspirilene, or fendiline may be at dosages, concentrations or schedules to achieve blood or plasma concentrations in a range of 1 -100, or 50-500, or 100-1000μg/L in persons on therapy with the drug. [0038] In certain instances, it will be desirable to have multiple administrations of the composition, e.g., 2, 3, 4, 5, 6 or more administrations. The administrations can be at 1, 2, 3, 4, 5, 6, 7, 8, to 5, 6, 7, 8, 9 ,10, 1 1 , 12 twelve day intervals, including all ranges there between. Additionally, the administrations can be at 1 , 2, 3, 4, 5, 6, 7, 8, to 5, 6, 7, 8, 9 ,10, 1 1 , 12 twelve week intervals, including all ranges there between. [00391 ^m y ^et °th ^er embodiments, a calcium cycling modulator inhibits calcium channel expression. Inhibition of calcium channel expression may be achieved by methods known to those of skill in the art. Particularly, RNA interference, antisense RNA, siRNA, gene editing are contemplated as methods to inhibit calcium channel expression. In still other aspects, a calcium cycling modulator inhibits calcium channel stability and leads to increased calcium channel turnover or degradation. In other aspects a calcium cycling modulator decreases the open probability of a calcium channel.

[0040] Nucleic acid molecules identical or complementary to all or part of any L-type calcium channel, ryanodine 2 receptor or calsequestrin 2 may be employed in diagnostic, prognostic and therapeutic methods and compositions described herein. [0041] In the disclosed methods, nucleic acids can be labeled, used as probes, in array analysis, or employed in other diagnostic or prognostic applications, particularly those related to detecting L-type calcium channel, ryanodine 2 receptor overexpression or calsequestrin 2 underexpression and/or related Fabry disease cardiac symptoms or manifestations. The expression of genes associated with Fabry disease cardiac symptoms or manifestations may be assayed or detected by methods used to detect and/or measure nucleic acid expression described below.

[0042] In addition nucleic acids can be used as antisense or siR A molecules targeted at a L-type calcium channel or ryanodine 2 receptor gene for use in reducing expression of that gene. In certain embodiments, reduction of expression provides inhibition of L-type calcium channel or ryanodine 2 receptor and accordingly activity of an L-type calcium channel or ryanodine 2 receptor. These therapeutic nucleic acids may be modified to enhance their stability in storage or in vivo, bioavailability, activity, or localization.

[0043] The nucleic acids may have been endogenously produced by a cell, or been synthesized or produced chemically or recombinantly. They may be isolated and/or purified. Nucleic acids used in methods and compositions disclosed herein may have regions of identity or complementarity to another nucleic acid, such as a L-type calcium channel or ryanodine 2 receptor. It is contemplated that the region of complementarity or identity can be at least 5 contiguous residues, though it is specifically contemplated that the region is, is at least, or is at most 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 441 , 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000, or any range derivable therein, contiguous nucleotides. It is further understood that the length of complementarity within a gene, gene transcript or between a gene target and a nucleic acid are such lengths. Moreover, the complementarity may be expressed as a percentage, meaning that the complementarity between a probe and its target is 90% or greater over the length of the probe. In some embodiments, complementarity is or is at least 90%, 95% or 100%, or any range derivable therein. In particular, such lengths may be applied to any nucleic acid comprising a nucleic acid sequence identified. The commonly used name of the genes or gene targets is given throughout the application. The sequence of a gene can be used to design the sequence of any probe, primer or siRNA molecule that is complementary or identical to a target L-type calcium channel or ryanodine 2 receptor gene identified herein.

[0044] It is understood that a nucleic acid may be derived from genomic sequences or a gene. In this respect, the term "gene" is used for simplicity to refer to the genomic sequence encoding the transcript for a given amino acid sequence. However, embodiments may involve genomic sequences of a gene that are involved in its expression, such as a promoter or other regulatory sequences.

[0045] The term "recombinant" generally refers to a molecule that has been manipulated in vitro or that is a replicated or expressed product of such a molecule. [0046] The term "nucleic acid" is well known in the art. A "nucleic acid" as used herein will generally refer to a molecule (one or more strands) of DNA, RNA or a derivative or analog thereof, comprising a nucleobase. A nucleobase includes, for example, a naturally occurring purine or pyrimidine base found in DNA (e.g., an adenine "A," a guanine "G," a thymine "T" or a cytosine "C") or RNA (e.g., an A, a G, an uracil "U" or a C). The term "nucleic acid" encompasses the terms "oligonucleotide" and "polynucleotide," each as a subgenus of the term "nucleic acid."

[0047] As used herein, "hybridization", "hybridizes" or "capable of hybridizing" is understood to mean the forming of a double or triple stranded molecule or a molecule with partial double or triple stranded nature. The term "anneal" is synonymous with "hybridize." The term "hybridization", "hybridize(s)" or "capable of hybridizing" encompasses the terms "stringent condition(s)" or "high stringency" and the terms "low stringency" or "low stringency condition(s)."

[0048] As used herein, "stringent condition(s)" or "high stringency" are those conditions that allow hybridization between or within one or more nucleic acid strand(s) containing complementary sequence(s), but preclude hybridization of random sequences. Stringent conditions tolerate little, if any, mismatch between a nucleic acid and a target strand. Such conditions are well known to those of ordinary skill in the art, and are preferred for applications requiring high selectivity. Non-limiting applications include isolating a nucleic acid, such as a gene or a nucleic acid segment thereof, or detecting at least one specific mRNA transcript or a nucleic acid segment thereof, and the like. [0049] Stringent conditions may comprise low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.5 M NaCl at temperatures of about 42°C to about 70°C. It is understood that the temperature and ionic strength of a desired stringency are determined in part by the length of the particular nucleic acid(s), the length and nucleobase content of the target sequence(s), the charge composition of the nucleic acid(s), and to the presence or concentration of formamide, tetramethylammonium chloride or other solvent(s) in a hybridization mixture.

[0050] It is also understood that these ranges, compositions and conditions for hybridization are mentioned by way of non-limiting examples only, and that the desired stringency for a particular hybridization reaction is often determined empirically by comparison to one or more positive or negative controls. Depending on the application envisioned it is preferred to employ varying conditions of hybridization to achieve varying degrees of selectivity of a nucleic acid towards a target sequence. In a non-limiting example, identification or isolation of a related target nucleic acid that does not hybridize to a nucleic acid under stringent conditions may be achieved by hybridization at low temperature and/or high ionic strength. Such conditions are termed "low stringency" or "low stringency conditions," and non-limiting examples of such include hybridization performed at about 0.15 M to about 0.9 M NaCl at a temperature range of about 20°C to about 50°C. Of course, it is within the skill of one in the art to further modify the low or high stringency conditions to suite a particular application. [0051 J A nucleic acid may comprise, or be composed entirely of, a derivative or analog of a nucleobase, a nucleobase linker moiety and/or backbone moiety that may be present in a naturally occurring nucleic acid. RNA with nucleic acid analogs may also be labeled according to methods disclosed herein. As used herein a "derivative" refers to a chemically modified or altered form of a naturally occurring molecule, while the terms "mimic" or "analog" refer to a molecule that may or may not structurally resemble a naturally occurring molecule or moiety, but possesses similar functions. As used herein, a "moiety" generally refers to a smaller chemical or molecular component of a larger chemical or molecular structure. Nucleobase, nucleoside, and nucleotide analogs or derivatives are well known in the art, and have been described (see for example, Scheit, 1980, incorporated herein by reference).

[0052] In some embodiments, nucleic acid molecules comprise at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, or at least 85% sequence complementarity to a target region within the target nucleic acid. In other embodiments, the molecules comprise at least 90% sequence complementarity to a target region within the target nucleic acid. In other embodiments, the molecules comprise at least 95% or at least 99% sequence complementarity to a target region within the target nucleic acid. For example, a nucleic acid molecule in which 18 of 20 nucleobases of it are complementary to a target sequence would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. As such, an oligomeric compound that is 18 nucleobases in length having 4 noncomplementary nucleobases that are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid. Percent complementarity of an oligomeric compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).

[0053] A nucleic acid molecule is "targeted" to a molecule (that is a nucleic acid) in some embodiments. This means there is sufficient sequence complementarity to achieve a level of hybridization that accomplishes a particular goal with respect to the nucleic acid molecule, such as in the context of being a probe, a primer or an siRNA. In some embodiments, expression or function is to be modulated. This targeted nucleic acid (or nucleic acid or gene target) may be, for example, a mRNA transcribed from a cellular gene whose expression is associated with a particular disorder or disease state, a small non-coding RNA or its precursor, or a nucleic acid molecule from an infectious agent.

[0054] The targeting process usually also includes determination of at least one target region, segment, or site within the target nucleic acid for the interaction to occur such that the desired effect, e.g., modulation of levels, expression or function, will result. Within the context of the present invention, the term "region" is defined as a portion of the target nucleic acid having at least one identifiable sequence, structure, function, or characteristic. Within regions of target nucleic acids are segments. "Segments" are defined as smaller or sub- portions of regions within a target nucleic acid. "Sites," as used in the present invention, are defined as specific positions within a target nucleic acid. The terms region, segment, and site can also be used to describe an oligomeric compound of the invention such as for example a gapped oligomeric compound having three separate segments.

[0055] Targets of the nucleic acid molecules described in embodiments include both coding and non-coding nucleic acid sequences. For coding nucleic acid sequences, the translation initiation codon is typically 5 -AUG (in transcribed mRNA molecules; 5 -ATG in the corresponding DNA molecule), the translation initiation codon is also referred to as the "AUG codon," the "start codon" or the "AUG start codon." A minority of genes have a translation initiation codon having the RNA sequence 5'-GUG, 5 -UUG or 5'-CUG, and 5'- AUA, 5'-ACG and 5'-CUG have been shown to function in vivo. Thus, the terms "translation initiation codon" and "start codon" can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine (in eukaryotes). It is also known in the art that eukaryotic and prokaryotic genes may have two or more alternative start codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions. In the context of the invention, "start codon" and "translation initiation codon" refer to the codon or codons that are used in vivo to initiate translation of an mRNA transcribed from a gene encoding a nucleic acid target, regardless of the sequence(s) of such codons. It is also known in the art that a translation termination codon (or "stop codon") of a gene may have one of three sequences, i.e., 5'-UAA, 5'-UAG and 5 -UGA (the corresponding DNA sequences are 5'- TAA, 5'-TAG and 5'-TGA, respectively). A. Preparation of Nucleic Acids

[0056] A nucleic acid may be made by any technique known to one of ordinary skill in the art, such as for example, chemical synthesis, enzymatic production, or biological production. It is specifically contemplated that nucleic acid probes are chemically synthesized. [0057] In some embodiments, nucleic acids are recovered or isolated from a biological sample. The nucleic acids may be recombinant or it may be natural or endogenous to the cell (produced from the cell's genome). It is contemplated that a biological sample may be treated in a way so as to enhance the recovery of small RNA molecules such as mRNA or miR As. U.S. Patent Application Serial No. 10/667,126 describes such methods and is specifically incorporated herein by reference. Generally, methods involve lysing cells with a solution having guanidinium and a detergent. [0058] Alternatively, nucleic acid synthesis is performed according to standard methods. See, for example, Itakura and Riggs (1980). Additionally, U.S. Patents 4,704,362, 5,221 ,619, and 5,583,013 each describe various methods of preparing synthetic nucleic acids. Non-limiting examples of a synthetic nucleic acid (e.g., a synthetic oligonucleotide) include a nucleic acid made by in vitro chemical synthesis using phosphotriester, phosphite, or phosphoramidite chemistry and solid phase techniques such as described in EP 266,032, incorporated herein by reference, or via deoxynucleoside H-phosphonate intermediates as described by Froehler et al., 1986 and U.S. Patent 5,705,629, each incorporated herein by reference. In some methods, one or more oligonucleotide may be used. Various different mechanisms of oligonucleotide synthesis have been disclosed in for example, U.S. Patents 4,659,774, 4,816,571 , 5, 141 ,813, 5,264,566, 4,959,463, 5,428,148, 5,554,744, 5,574,146, 5,602,244, each of which is incorporated herein by reference.

[0059] A non-limiting example of an enzymatically produced nucleic acid include one produced by enzymes in amplification reactions such as PCR™ (see for example, U.S. Patents 4,683,202 and 4,682,195, each incorporated herein by reference), or the synthesis of an oligonucleotide as described in U.S. Patent 5,645,897, incorporated herein by reference. A non-limiting example of a biologically produced nucleic acid includes a recombinant nucleic acid produced (i.e., replicated) in a living cell, such as a recombinant DNA vector replicated in bacteria (see for example, Sambrook et al., 2001, incorporated herein by reference). [0060] Oligonucleotide synthesis is well known to those of skill in the art. Various different mechanisms of oligonucleotide synthesis have been disclosed in for example, U.S. Patents 4,659,774, 4,816,571 , 5,141 ,813, 5,264,566, 4,959,463, 5,428,148, 5,554,744, 5,574,146, 5,602,244, each of which is incorporated herein by reference.

[0061] Basically, chemical synthesis can be achieved by the diester method, the triester method, polynucleotide phosphorylase method, and by solid-phase chemistry. The diester method was the first to be developed to a usable state, primarily by horana and co- workers. ( horana, 1979). The basic step is the joining of two suitably protected deoxynucleotides to form a dideoxynucleotide containing a phosphodiester bond.

[0062] The main difference between the diester and triester methods is the presence in the latter of an extra protecting group on the phosphate atoms of the reactants and products (Itakura et al., 1975). Purifications are typically done in chloroform solutions. Other improvements in the method include (i) the block coupling of trimers and larger oligomers, (ii) the extensive use of high-performance liquid chromatography for the purification of both intermediate and final products, and (iii) solid-phase synthesis.

[0063] Polynucleotide phosphorylase method is an enzymatic method of DNA synthesis that can be used to synthesize many useful oligonucleotides (Gillam et al., 1978; Gillam et al., 1979). Under controlled conditions, polynucleotide phosphorylase adds predominantly a single nucleotide to a short oligonucleotide. Chromatographic purification allows the desired single adduct to be obtained. At least a trimer is required to start the procedure, and this primer must be obtained by some other method. The polynucleotide phosphorylase method works and has the advantage that the procedures involved are familiar to most biochemists.

[0064] Solid-phase methods draw on technology developed for the solid-phase synthesis of polypeptides. It has been possible to attach the initial nucleotide to solid support material and proceed with the stepwise addition of nucleotides. All mixing and washing steps are simplified, and the procedure becomes amenable to automation. These syntheses are now routinely carried out using automatic nucleic acid synthesizers.

[0065] Phosphoramidite chemistry (Beaucage and Lyer, 1992) has become the most widely used coupling chemistry for the synthesis of oligonucleotides. Phosphoramidite synthesis of oligonucleotides involves activation of nucleoside phosphoramidite monomer precursors by reaction with an activating agent to form activated intermediates, followed by sequential addition of the activated intermediates to the growing oligonucleotide chain (generally anchored at one end to a suitable solid support) to form the oligonucleotide product.

[0066] Recombinant methods for producing nucleic acids in a cell are well known to those of skill in the art. These include the use of vectors (viral and non-viral), plasmids, cosmids, and other vehicles for delivering a nucleic acid to a cell, which may be the target cell (e.g., a cardiac cell) or simply a host cell (to produce large quantities of the desired RNA molecule). Alternatively, such vehicles can be used in the context of a cell free system so long as the reagents for generating the RNA molecule are present. Such methods include those described in Sambrook, 2003, Sambrook, 2001 and Sambrook, 1989, which are hereby incorporated by reference.

[0067] In certain embodiments, nucleic acid molecules are not synthetic. In some embodiments, the nucleic acid molecule has a chemical structure of a naturally occurring nucleic acid and a sequence of a naturally occurring nucleic acid. In addition to the use of recombinant technology, such non-synthetic nucleic acids may be generated chemically, such as by employing technology used for creating oligonucleotides.

B. Isolation of Nucleic Acids

[0068] Nucleic acids may be isolated using techniques well known to those of skill in the art, though in particular embodiments, methods for isolating small nucleic acid molecules, and/or isolating mRNA molecules can be employed. Chromatography is a process often used to separate or isolate nucleic acids from protein or from other nucleic acids. Such methods can involve electrophoresis with a gel matrix, filter columns, alcohol precipitation, and/or other chromatography. If mRNA from cells is to be used or evaluated, methods generally involve lysing the cells with a chaotropic (e.g., guanidinium isothiocyanate) and/or detergent (e.g., N-lauroyl sarcosine) prior to implementing processes for isolating particular populations of RNA.

[0069] In particular methods for separating mRNA from other nucleic acids, a gel matrix is prepared using polyacrylamide, though agarose can also be used. The gels may be graded by concentration or they may be uniform. Plates or tubing can be used to hold the gel matrix for electrophoresis. Usually one-dimensional electrophoresis is employed for the separation of nucleic acids. Plates are used to prepare a slab gel, while the tubing (glass or rubber, typically) can be used to prepare a tube gel. The phrase "tube electrophoresis" refers to the use of a tube or tubing, instead of plates, to form the gel. Materials for implementing tube electrophoresis can be readily prepared by a person of skill in the art or purchased.

[0070] Methods may involve the use of organic solvents and/or alcohol to isolate nucleic acids, particularly RNA used in methods and compositions disclosed herein. Some embodiments are described in U.S. Patent Application Serial No. 10/667,126, which is hereby incorporated by reference. Generally, this disclosure provides methods for efficiently isolating small RNA molecules from cells comprising: adding an alcohol solution to a cell lysate and applying the alcohol/lysate mixture to a solid support before eluting the RNA molecules from the solid support. In some embodiments, the amount of alcohol added to a cell lysate achieves an alcohol concentration of about 55% to 60%. While different alcohols can be employed, ethanol works well. A solid support may be any structure, and it includes beads, filters, and columns, which may include a mineral or polymer support with electronegative groups. A glass fiber filter or column may work particularly well for such isolation procedures. [0071] In specific embodiments, RNA isolation processes include: a) lysing cells in the sample with a lysing solution comprising guanidinium, wherein a lysate with a concentration of at least about 1 M guanidinium is produced; b) extracting RNA molecules from the lysate with an extraction solution comprising phenol; c) adding to the lysate an alcohol solution for forming a lysate/alcohol mixture, wherein the concentration of alcohol in the mixture is between about 35% to about 70%; d) applying the lysate/alcohol mixture to a solid support; e) eluting the RNA molecules from the solid support with an ionic solution; and, f) capturing the RNA molecules. Typically the sample is dried down and resuspended in a liquid and volume appropriate for subsequent manipulation.

[0072] Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, are double-stranded RNA molecules, 20-25 base pairs in length. siRNAs form part of the RNA interference (RNAi) pathway, where they interfere with the expression of specific genes with complementary nucleotide sequence. Any of the embodiments discussed above regarding nucleic acids may be implemented with respect to siRNA molecules. [0073] Therapeutic siRNA may be administered to a patient to modulate the expression of one or more of the genes identified as involved in calcium homeostasis in cardiac cells related to Fabry disease. Therapeutic siRNA may also target the expression of the spectrum of genes/gene products related to calcium homeostasis (e.g., L-type calcium channels, ryanodine 2 receptor). The design of siRNA to target expression of a gene is a process well known in the art (Nat Biotechnol. 2004 Mar;22(3):326-30. Rational siRNA design for RNA interference. Reynolds A, Leake D, Boese Q, Scaringe S, Marshall WS, hvorova A.). Design of siRNA to target any of the genes identified in cardiac calcium homeostasis may be done using the publicly available sequences (UniProt Q92736: Ryanodine receptor 2; UniProt Q 13936, 060840, Q 13698, Q01668, Q02641 , Q08289, P54284, 000305, Q59GD8, B7Z269, B1ALM3, F5H522, E9PDI6, F5GY28, F5H638, F8WA06, F5H313, A6PVM6, F5H0X0, F8VW 1 , E7E 1 1, C9J224, F8VNV8, H0Y476, B7Z658, H7C4S8, F8VV14, F8VU10, F8VUW8, Q5VVH1 , F8W0F8, H0YHK1 , B7Z4Q4, Q59GU3, B7Z1U5, B7Z3Y0, H7C549, H0YHY2, Q5V9X9, B7Z1S2, B4DGG5, B7Z7Z5, F8W1N3, B7Z6T5, Q86XX0, Q86XX1 , Q5V9X8: L-type calcium channel).

[0074] In certain embodiments the therapeutic siRNA modulates an L-type calcium channel gene selected from CACNA1 C, CACH2, CACN2, CACNL1 A1 , CCHL1A1 , CACNA1C, CACH2, CACN2, CACNL1 A1 , CCHL1 A1 , CACNA1F, CACNAF1, CACNA1 S, CACH1, CACN1 , CACNL1A3, CACNA1 D, CACH3, CACN4, CACNL1 A2, CCHL1A2, CACNB1 CACNLB1 , CACNB2, CACNLB2, MYSB, CACNB3, CACNLB3, CACNB4, CACNLB4, CACNA1 D, CACNA1D, CACNA1 S, CACNA1 C, CACNA1C, CACNA1C, CACNA1C, CACNB4, CACNA1D, CACNB2, CACNA1 C, CACNB3, CACNB4, CACNB4, CACNB3, CACNB4, CACNA2D1 , CACNA1 D, CACNB3, CACNB3, CACNB3, CACNB3, CACNB3, CACNA1F, CACNB3, CACNA1C, CACNB3, CACNA1 C, CACNA1C, CACNA1C.

[0075] The generation of siRNA molecules may, but is not limited to, methodology described in paragraphs above. In addition, the nucleic acid molecules described above may be employed as siRNA molecules targeting a L-type calcium channel or ryanodine receptor such as ryanodine receptor 2.

(0076] In certain aspects a calcium cycling modulator targets a ryanodine receptor. In certain aspects, a calcium cycling modulator targets ryanodine receptor 2. Ryanodine receptor 2 expression or activity may be targeted by therapeutic siRNA or therapeutic molecules that direct the knockdown of ryanodine receptor 2 through the RNA interference pathway. Ryanodine receptor 2 activity may be therapeutically modulated by altering its phosphorylation state by drugs and phosphatases that generally or specifically target ryanodine receptor 2. Additionally, drugs or pharmacological modulators that decrease ryanodine receptor 2 open probability may be used to therapeutically inhibit ryanodine receptor 2 activity. Pharmacological modulators may either stimulate or inhibit Ca2+ release, depending on concentration or incubation time, such concentration and exposure times are known to those of ordinary skill in the art. Ryanodine receptor 2 pharmacological modulators include but are not limited to ryanoids, purine derivatives and related compounds, methylxanthines, carboline derivatives and carbazole derivatives, sulmazole, anthraquinones, digitalis glycosides, milrinone and other bipyridine derivatives, suramin, halogenated hydrocarbons and phenols, volatile anesthetics, phenol derivatives, hexachlorocyclohexane. macrocyclic compounds immunosuppressant macrolides, bastadins, quinolidomicin al, heparin polyamines, ruthenium red, aminoglycosides, fla365, dantrolene, local anesthetics and phenylalkylamines.

[0077] Certain embodiments involve a calcium cycling modulator that is 1 ,4- benzothiazepine analogue JTV519 (K201), JTV519 analogue (SI 07), RyR2 blocking agent tetracaine or CaMK.II inhibitor KN-93. In some embodiments, the calcium cycling modulator is BAPTA or EDTA.

[0078] In certain embodiments calcium homeostasis is treated in cardiac cell of a Fabry disease subject by increasing calcium buffering. In certain aspects the activity of calsequestrin-2 is increased or potentiated. General Pharmaceutical Compositions

[0079] In some embodiments, pharmaceutical compositions are administered to a subject. Different aspects may involve administering an effective amount of a composition to a subject. In some embodiments, a composition comprising a calcium cycling modulator may be administered to the patient to treat Fabry disease. More specifically, regulation or homeostatsis of calcium treats cardiac manifestations associated with Fabry disease, including arrhythmias, cardiac hypertrophy and bradychardia. Alternatively, in the case of compositions comprising nucleic acid or proteinaceous effectors, an expression vector encoding one or more such nucleic acids, polypeptides or peptides may be given to a patient as a treatment. Additionally, such compositions can be administered in combination with traditional cardiac therapies to treat arrhythmias, cardiac hypertrophy and bradychardia. Such compositions will generally be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

[0080] The phrases "pharmaceutically acceptable" or "pharmacologically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal or human. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients, its use in immunogenic and therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

[0081] The active compounds can be formulated for parenteral administration, e.g., formulated for injection via the mucosal, intravenous, intramuscular, sub-cutaneous, or even intraperitoneal routes. Typically, such compositions can be prepared as either liquid solutions or suspensions; solid forms suitable for use to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and, the preparations can also be emulsified.

[0082] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that it may be easily injected. It also should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

[0083] A proteinaceous compositions may be formulated into a neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

[0084] A pharmaceutical composition can include a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0085] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization or an equivalent procedure. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum- drying and freeze-drying techniques, which yield a powder of the active ingredient, plus any additional desired ingredient from a previously sterile-filtered solution thereof. [0086] Administration of the compositions will typically be via any common route.

This includes, but is not limited to oral, nasal, or buccal administration. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, intranasal, or intravenous injection. Such compositions would normally be administered as pharmaceutically acceptable compositions that include physiologically acceptable carriers, buffers or other excipients.

[0087] An effective amount of therapeutic or prophylactic composition is determined based on the intended goal. The term "unit dose" or "dosage" refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the composition calculated to produce the desired responses discussed above in association with its administration, i.e., the appropriate route and regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the protection desired.

[0088] Precise amounts of the composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the subject, route of administration, intended goal of treatment (alleviation of symptoms versus cure), and potency, stability, and toxicity of the particular composition. [0089 J Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above. (0090) Administration of drugs such as Verapamil, Gallopamil, Fendiline, Diltiazem, mibefradil, bepridil, fluspirilene, or fendiline may be at dosages, concentrations or schedules to achieve blood or plasma concentrations in a range of 1-100, or 50-500, or 100-1000μg/L in persons on therapy with the drug.

[0091] In certain instances, it will be desirable to have multiple administrations of the composition, e.g., 2, 3, 4, 5, 6 or more administrations. The administrations can be at 1 , 2, 3, 4, 5, 6, 7, 8, to 5, 6, 7, 8, 9 ,10, 1 1 , 12 twelve day intervals, including all ranges there between. Additionally, the administrations can be at 1 , 2, 3, 4, 5, 6, 7, 8, to 5, 6, 7, 8, 9 ,10, 1 1 , 12 twelve week intervals, including all ranges there between.

Combination Therapy

[0092] The compositions and related methods, particularly administration of a therapy to restore calcium homeostasis that comprises inhibitors of L-type calcium channel or ryanodine receptor 2 expression or activity, a potentiator of PLN or CASQ2 activity, a RyR2 specific or general phosphatase, Ryr2 stabilizing agent may also be used in combination with the administration of Fabry disease enzyme replacement therapy (alpha-galctosidase A therapy or an enzyme that mimics alpha-galactosidase A activity such as agalsidase beta, also known as Fabryzme ®) or traditional therapies or drugs to treat arrhythmias, bradychardia, cardiac hypertrophy or other cardiac manifestations of Fabry disease. These include but are not limited to Amiodarone (Cordarone, Pacerone), Bepridil Hydrochloride (Vascor), Disopyramide (Norpace), Dofetilide (Tikosyn), Dronedarone (Multaq), Flecainide (Tambocor), Ibutilide (Corvert), Lidocaine (Xylocaine), Procainamide (Procan, Procanbid), Propafenone (Rythmol), Propranolol (Inderal), Quinidine (many trade names), Sotalol (Betapace), and Tocainide (Tonocarid). An arrhythmia therapy may comprise any form of ablation, defibrillation or a device to treat arrhythmia. Forms of ablation include radiofrequency ablation, transcatheter ablation or catheter ablation. Devices contemplated to treat arrhythmias include implantable cardioverter defibrillators and pacemakers. A treatment of cardiac hypertrophy may specifically treat ventricular hypertrophy, left ventricular hypertrophy, right ventricular hypertrophy or associate hypertension. Examples of medications that may be used to treat ventricular hypertrophy an associated hypertension include but are not limited to Cozaar®(losartan potassium) and Hyzaar®(Losartan- hydrochlorothiazide). Treatments for bradychardia may include epinephrine, dopamine, atropine, hyoscyamine sulfate, or Levsin®. [0093] In one aspect, it is contemplated that a therapy to restore calcium homeostasis includes inhibitors of L-type calcium channel or ryanodine receptor 2 expression or activity, potentiator of PLN or CASQ2 activity, RyR2 specific or general phosphatase, Ryr2 stabilizing agent that are used in conjunction with Fabry disease enzyme replacement therapy (alpha-galctosidase A therapy or an enzyme that mimics alpha-galactosidase A activity such as agalsidase beta, also known as Fabryzme ®) or traditional therapies or drugs to treat arrhythmias, bradychardia, cardiac hypertrophy or other cardiac manifestations of Fabry disease. Alternatively, the therapy may precede or follow the other agent treatment by intervals ranging from minutes to weeks. In embodiments where the other agents, drugs and/or a proteins or polynucleotides are administered separately, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the therapeutic composition would still be able to exert an advantageously combined effect on the subject. In such instances, it is contemplated that one may administer both modalities within about 12-24 h of each other and, more preferably, within about 6-12 h of each other. In some situations, it may be desirable to extend the time period for administration significantly, however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations.

(0094] Various combinations of therapy may be employed, for example inhibitors of L-type calcium channel or RyR2 expression or activity, potentiator of PLN or CASQ2 activity, RyR2 specific or general phosphatase, Ryr2 stabilizing agent is "A" and traditional therapies or drugs to treat arrhythmias, bradychardia, cardiac hypertrophy or other cardiac manifestations of Fabry disease is "B":

A/B/A B/A/B B B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B

B/B/B/A B/B/A/B A/ A/B B A/B/A/B A/B/B/A B/B/A/A

B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A [0095] Administration of the antibody compositions to a patient/subject will follow general protocols for the administration of such compounds, taking into account the toxicity, if any, of the composition. It is expected that the treatment cycles would be repeated as necessary. It is also contemplated that various standard therapies, such as hydration, may be applied in combination with the described therapy.

IV. Examples

[0096] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. Example 1

[0097] Fabry disease is an X-linked lyosomal storage disorder, caused by insufficient activity of a-galactosidase A. As a result of enzyme deficiency glycosphingolipids with terminal a-D-galactosyl moieties, primarily globotriaosylceramide, accumulate in multiple organs. Arrhythmias and cardiac hypertrophy are the most prominent cardiac manifestations of Fabry disease, which can cause sudden death and precipitate heart failure in the patients. The pathogenesis of arrhythmias in Fabry disease remains unclear.

(0098] The major obstacle in studying the pathogenesis of Fabry heart disease at cellular and molecular level is the shortage of available human cardiac cells from the patients for research purpose. To overcome this problem, the inventors generated induced pluripotent stem cells (iPSC) from skin fibroblasts of Fabry male hemizygous patients (total deficiency in a-Gal A activity) and age-matched healthy controls. Patient iPSC were differentiated into functional, spontaneously contracting cardiomyocytes.

[0099] Methods - Induced pluripotent stem cells (iPSC) from skin fibroblasts of Fabry male hemizygous patients (total deficiency in a-galactosidase A activity) and age- matched health controls were generated by VSV.g-pseudotyped Moloney-based retroviral transduction of human Sox2, Oct3/4, c-myc and lf-4. The iPSCs were differentiated to spontaneously contracting cardiomyocytes by embryoid body-based method. Disease phenotypes were characterized in Fabry patient cardiomyocytes. The expression of major ion channels and calcium regulatory proteins were analyzed by quantitative RT-PCR and compared between the cardiomyocytes from Fabry patient and healthy controls.

[00100] The patient iPSC-derived cardiac cells recaptured the arrhythmia disease phenotype in vitro. The inventors found that Fabry cardiomyocytes have slower beating rates than healthy controls, mimicking the clinical bradycardia that is present in these donor Fabry patients. Treatment of Fabry cardiomyocytes with recombinant a-Gal A significantly increased the beating rates compared with mock-treated counterparts. This suggested that arrhythmia in Fabry disease is likely caused by intrinsic dysfunction of cardiomyocytes that iPSC-derived cardiomyocytes are a useful model for studying disease mechanism of arrhythmia in Fabry disease. By analyzing expression levels of major ion channels and Ca2+ handling proteins in iPSC-derived cardiomyocytes, the inventors found the expression levels of 2 molecules that play fundamental roles in Ca2+ homeostasis are significantly abnormal in Fabry patients' cardiomyocytes: increased L-type calcium channel (LTCC) and decreased calsequenstrin 2 (CASQ2).

[00101] Generation of iPSC-derived Fabry patients cardiomyocytes. The inventors generated iPSCs from dermal fibroblasts of Fabry patients (male hemizygous, n=2) and healthy controls (male, n=2) by VSV.g-pseudotyped Moloney-based retroviral transduction of human Sox2, Oct3/4, c-myc and Klf-4. Established iPSC clones are pluripotent by the assessment of their expression of pluripotent stem cell markers (Fig. 1) and teratoma formation assay (data not shown).

[00102] Arryhthmia phenotype in iPSC-derived Fabry patient cardiomyocytes. Fabry patient and healthy control iPSC were differentiated into spontaneously contracting cardiomyocytes in vitro by embryoid body (EB)-based cardiac differentiation method [4]. The spontaneous beating rates of cardiac clusters were measured and compared between Fabry patients and healthy controls at day 30 of differentiation. The beating rates in the Fabry patient cardiomyocytes (16.7 ± 1.04 beat/min, n=41 and 21.6 ± 2.84 beat min, n=19) are significantly lower compared with healthy controls (49.1 ± 8.51 beat/min, n=17 and 46.3 ± 5.61 beat/min, n=21). (Fig. 4) Abnormal expression of calcium handling proteins

[00103] Up-regulated L-type calcium channel (LTCC) expression. The electrical activities (action potential) of cardiomyocytes are mediated by the ion flows across the cell membranes. Ion channels are proteins responsible for the gating of these ion flows. The expression and function of these channel proteins is important for maintaining the normal electrical activities of cardiomyocytes. The inventors studied the expression levels of major ion channels in iPSC-derived cardiomyocytes by quantitative RT-PCR and compared between Fabry patients and healthy controls. The inventors found the expression of LTCC, the cell membrane sensor the calcium cycling (Wehrens, et al, 2004), is significantly up- regulated in Fabry disease patient cardiomyocytes (Figure 6).

[00104] The inventors further studied whether there is abnormal expression of other calcium handling proteins. The inventors found significant down-regulated expression of CASQ2 at mRNA level, the most abundant calcium buffering protein in cardiomyocytes [6]. [00105] Treatment of arrhythmias. Calcium handling abnormalities play a central role in the pathogenesis of a variety of heart conditions including arrhythmias, cardiomyopathies and heart failure (Yano, et al., 2008; Tomaselli, 2012). Targeting the molecules/pathways in the calcium cycling can be a novel effective treatment strategies for the arrhythmia in Fabry disease. [00106] Target molecules that can reduce calcium overload in Fabry cardiac cells.

These include the of LTCC inhibitors such as Diltiazem, verapamil. The use may be before cardiac arrhythmias occur.

[00107] Target molecules that prevent/reduce calcium leak in Fabry cardiac cells. These include RyR2 stabilizing molecules such as JTV-519 (Aetas Pharma Ltd. Japan) and/or over-expression of calcium buffering protein or drug of the sarcoplasmic reticulum such as CASQ2 by gene transfer.

[00108] Treatment to other cardiac complications. Abnormal calcium cycling could also be the potentially relevant to the pathogenesis of other cardiac manifestations in Fabry disease. Fabry patients chronically develop left ventricle hypertrophy and sometimes followed by a congestive heart failure. Approaches that can restore the normal calcium homeostasis in the early stage of the disease can be the effective preventive/ therapeutic treatment to these cardiac abnormalities.

[00109] Treatment to other organ systems. Besides muscle contraction calcium cycling also participates in crucial events in the cells such as proliferation, secretion, apoptosis and excitotoxicity. The disturbance in calcium cycling can also potentially contribute to pathogenesis of Fabry disease of other organ systems and cell types other than the heart and cardiomyocytes. Therefore approaches that targeting the molecules/pathways in calcium cycling can also be a potential treatment strategies for the manifestation of these organs/cells in Fabry disease patients. [00110] Conclusions - Fabry patient iPSC-derived cardiomyocytes recaptured arrhythmias phenotype in vitro; The expression of several calcium regulatory proteins are significantly abnormal in Fabry patient cardiomyocytes.

Example 2

Abnormal Ca2+ handling in Fabry patient iPSC-derived cardiomyocytes [00111] To study the abnormalities in Ca2+ handling in Fabry disease, Ca2+ imaging studies were performed. To identify ventricular cardiomyocytes, Fabry patient and healthy control iPSC-derived cardiomyocytes were transduced with a Lentiviral vector (LV- MLC2v-tdTomato). Tomato-red positive ventricular cardiomyocytes were identified and loaded with Ca2+ sensitive dye Fluo-3 (Fig. 7A). The Ca2+ transients in Fabry patients and control cardiomyocytes were studied (Fig. 7B).

[00112] The spontaneous firing rates in Fabry patients' cardiomyocytes were significantly lower than those in healthy controls (Fig. 7C), indicating that the "bradycardia" of the cardiomyocytes observed in the Fabry patient cardiac clusters is also present at the single cell level. There was no significant difference in the amplitude of the Ca2+ transients between patients' and controls' cardiomyocytes. However, Fabry patients' cardiomyocytes had decreased upstroke velocity and increased decay velocity compared with healthy controls' cells (Fig. 7C). This can be associated with increased Ca2+ release from sarcoplasmic reticulum (SR.) to the cytosol and potentially cause intracellular Ca2+ overload in Fabry disease cardiomyocytes. Hyperphosphorylation of RyR2 and decreased phospholamban (PLN) protein expression in Fabry disease cardiac cells

[00113] The activities of RyR2 and SERCA2a receptors regulate SR Ca2+ release and uptake. In order to know whether there are abnormalities in the activity of these receptors that might contribute to the abnormalities in Ca2+ release and uptake, the inventors studied the protein expression levels of RyR2 receptor, SERCA2a and PLN in Fabry patient and healthy control iPSC-derived cardiac clusters by Western blot.

[00114] There were no significant changes in the total protein expressions of RyR2 (Fig. 8A) and SERCA2a (data not shown). However, there was increased phosphorylation of RyR2 at S2808 in Fabry patient iPSC-derived cardiac cells (Fig. 8 A). Hyperphosphorylation of RyR2 can lead to increased open channel probability , generating diastole Ca2+ leak in arrhythmia(Yano et al). This finding could be the molecular basis for the abnormal Ca2+ release in Fabry disease cells. Since the total protein level of SERCA2a did not change significantly, the inventors analysed protein expression and phosphorylation state of phospholamban (PLN), a key regulator of SR Ca2+ cycling through its inhibitory effect on the affinity of SERCA2a to Ca2+ The total PLN level is lower in Fabry patient cardiac cells compared with controls (Fig 8A). There was no change in phosphorylated PLN-to-total PLN ratios. This indicates that SERCA2a activity might be less inhibited by PLN in Fabry disease, which probably can lead to increased Ca2+ uptake into SR. In order to confirm that the molecular changes in the patient iPSC-derived cardiac cells also exist in vivo, the inventors analysed the protein expression levels of RyR2, SERCA2a and PLN in Fabry mouse heart tissues (Fig. 8B). Consistent with the human cells data, hyperphosphorylation of RyR2 and decreased PLN total protein was observed in heart tissues of 5 months old Fabry mice (Fig. 8B). LTCC inhibitor, verapamil improved "bradycardia " in Fabry patient iPSC-derived cardiomyocytes

[00115] The inventors further studied whether targeting abnormal Ca2+ cycling can correct bradycardia in Fabry disease. LTCC is the main entrance for Ca2+ influx in cardiac cells and determines the activity of the whole heart. Changes in Ca2+ influx balance in the cardiac cells are directly related to human and animal cardiac diseases, including arrhythmia and cardiac hypertrophy (Mukherjee et al.). The inventors observed increased mRNA expression of LTCC in Fabry patient-derived cardiac cells and heart tissues of Fabry mice, treated Fabry patients' cardiac clusters with a LTCC inhibitor, verapamil, and evaluated its effect on decreasing beating rates. At higher doses verapamil decreased beating rates in both Fabry patients' and healthy controls' cells (Fig. 9b). However, at a low dose (10e-9 M) the inhibitor significantly increased the beating rates of Fabry cardiomyocytes. The beating rate of normal control cells remained unchanged at these low verapamil concentrations. (Fig. 9).

[00116] In conclusion, the inventors identified abnormal SR Ca2+ release and uptake, upregulated LTCC expression, hyperphosphorylation of RyR2, decreased total PLN in Fabry patient iPSC-derived cardiomyocytes. These findings suggest that abnormal Ca2+ handling underlies the pathogenesis of Fabry heart disease and is a therapeutic target for treatment of arrhythmia in Fabry disease. LTCC inhibitor, verapamil improved beating rates of Fabry patient-derived cardiac clusters, suggesting that targeting Ca2+ cycling is effective in improving arrhythmia in Fabry disease and possibly other Fabry cardiac manifestations such as hypertrophic cardiomyopathy and vascular disease.

[00117] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. REFERENCES

The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

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